There is increasing evidence that lymphocytes can target and inhibit myeloid leukemia cells both in the allogeneic and autologous settings. The potent graft versus leukemia (GVL) effect associated with allogeneic stem cell transplant (SCT) conveys activity against leukemia and contributes to the decreased relapse risk compared with patients who do not receive an allogeneic graft. While GVL can induce long-term remission, the potentially lethal complication of graft-versus-host disease (GVHD) limits overall effectiveness of this treatment approach. We hypothesize that GVL would be enhanced, and GVHD reduced or eliminated, if the target antigens that drove those responses were identified, and if T cells with GVL antigen specificity could be isolated and adoptively transferred to patients with leukemia in the allogeneic setting, and that this effect could be increased by vaccination with appropriate leukemia antigens. We have identified an HLA-A2-restricted nonomer peptide, PR1, as a target antigen of CTL that inhibits myeloid leukemia progenitors and kills myeloid leukemia bone marrow cells. We have found that PR1 is processed from both proteinase 3 (PRTN3) and neutrophil elastase (ELA2), which are aberrantly expressed in leukemia. After vaccination with PR1, immune responses were observed in the majority of patients, and persistent remission was documented in some patients with AML, CML, and MDS. Immune-based therapies with low toxicity could be useful in combined treatment strategies to induce remission in patients with leukemia in the allogeneic setting to enhance GVL and minimize GVHD. In this proposal, we will (1) determine whether phenotype, function, and T cell receptor (TCR) affinity differ between PR1-CTL elicited after vaccination compared to those derived from healthy donors;(2) determine whether aberrant trafficking of PRTN3 and ELA2 can reverse tolerance and facilitate the expansion of central memory PR1-CTL;and (3) determine whether PR1-CTL expressing high affinity TCR can be transferred to patients with AML after T cell-depleted haplo-identical SCT to boost GVL and diminish GVHD. Lay Description: Stem cell transplantation has been used to successfully treat patients with leukemia, but the complication of graft-versus-host disease and the limited availability of suitable stem cell donors limit our use of this otherwise life-saving treatment to less than 25% of the potential patients who could benefit. To improve the effectiveness of SCT, we have identified some of the leukemia-associated target molecules recognized by the donor lymphocytes that mediate an immune effect against the patient's leukemia. We used one of these antigens, PR1, as a vaccine to boost immunity against leukemia in some patients with refractory disease. We found that PR1 vaccination was associated with little toxicity, and it boosted immunity in the majority of patients and induced remission in some patients, which has lasted up to seven years. In this proposal, we will study the molecular basis for immunity to this target, and we will use a strategy identified during the previous grant period to expand donor lymphocytes against this antigen, that we then transfer to patients with leukemia. If successful, this technique could eventually be used to reduce GVHD while retaining the beneficial effect of long-term remission after SCT.